The role of endothelin in mediating virus-induced changes in endothelin
The role of endothelin in mediating virus-induced changes in endothelin
Copyright #ERS Journals Ltd 1999 European Respiratory Journal ISSN 0903-1936 Eur Respir J 1999; 14: 92±97 Printed in UK ± all rights reserved The role of endothelin in mediating virus-induced changes in endothelinB receptor density in mouse airways P.J. Henry*, M.J. Carr*, R.G. Goldie*, A.Y. Jeng** The role of endothelin in mediating virus-induced changes in endothelinB receptor density in mouse airways. P.J. Henry, M.J. Carr, R.G. Goldie, A.Y. Jeng. #ERS Journals Ltd 1999. ABSTRACT: Emerging evidence supports a mediator role for endothelin (ET)-1 in airway diseases including asthma. Respiratory tract viral infections, are associated with increased levels of ET and altered ET receptor density and function in murine airways. To determine whether these virus-induced effects are causally linked, perhaps involving ET-1-induced ETB receptor downregulation, the current study investigated the influence of in vivo administration of CGS 26303, an ET-converting enzyme inhibitor, on virus-induced changes in ET-content and ETB receptor density. CGS 26303 (5 mg.kg-1.day-1) or placebo was administered to mice via osmotic minipumps implanted subcutaneously. Two days after implantation, mice were inoculated with influenza A/PR-8/34 virus or sham-infected, and all measurements were performed on tissue obtained on the fourth day post-inoculation. Viral infection was associated with elevated levels of immunoreactive ET and decreased densities of ETB receptors in murine airways. Both of these effects were attenuated in virus-infected mice that had received CGS 26303. Virus-induced increases in wet lung weight were also inhibited by CGS 26303. Importantly, administration of CGS 26303 had no effect on the titres of infectious virus in the lungs and similarly, viral infection had no effect on the plasma levels of free CGS 26303. In summary, CGS 26303 inhibited the virus-induced changes in both immunoreactive endothelin content and endothelinB receptor density. These findings are consistent with the postulate that the elevated epithelial expression of endothelin-1 during respiratory tract viral infection is a contributing factor in the downregulation of endothelinB receptors in airway smooth muscle. Whether inhibitors of endothelin synthesis attenuate virus-induced exacerbations of asthma or airways hyperresponsiveness remains to be established. Eur Respir J 1999; 14: 92±97. The levels of endothelin (ET)-1 in the airways are significantly elevated in respiratory diseases such as asthma  as well as in several animal models of airways disease, including allergic inflammation [2, 3] and respiratory tract viral infection . ET-1, via its potent actions on a raft of different cell types within the airways and lungs (for review see ), may contribute significantly to airway wall remodelling, oedema, bronchial obstruction, long-lasting bronchoconstriction and the development of airway hyperresponsiveness in asthma and during respiratory tract viral infections. Respiratory syncitial virus induces the expression of ET-1 in bronchial epithelial cells  and the levels of immunoreactive (ir)-ET-1 are elevated in murine airways and lung during influenza A viral infection . Increases in the production of ET-1 by the airway epithelium would be expected to lead to enhanced stimulation of ETA and ETB receptors in adjacent tissues such as the airway smooth muscle. However, respiratory tract viral infection in mice was associated with reductions in ETB receptor density, which was reflected in attenuated ETB receptormediated contractile function [7, 8]. This raises the intriguing possibility that the enhanced levels of ir-ET *Dept of Pharmacology, University of Western Australia, Australia. **Novartis Institute for Biomedical Research, Summit, New Jersey, USA. Correspondence: P.J. Henry Dept of Pharmacology University of Western Australia Nedlands, 6907 Western Australia Australia Fax: 61 893463469 Keywords: Endothelin-1 endothelin-converting enzyme inhibitors endothelin receptors influenza A virus lung tracheal smooth muscle Received: January 27 1999 Accepted after revision March 29 1999 This study was supported by the National Health and Medical Research Council (NH&MRC) of Australia, the Asthma Foundation of Western Australia, the Medical Fund of Western Australia and the Raine Foundation. contribute to the reduction in ETB receptor density and function, perhaps associated with ET-induced ETB receptor downregulation. In support of this postulate, ETB receptors present in murine and rat tracheal smooth muscle were readily desensitized in vitro . To further test the link between virus-induced increases in ir-ET and reductions in ETB receptor density, mice in the current study were treated with an ET-converting enzyme (ECE) inhibitor CGS 26303 to attenuate virus-induced increases in ET. If the postulate holds, then administration of CGS 26303 should be associated with inhibition of virusinduced changes in ETB receptor density as well as ET content. Methods Drug administration Eight-week-old male CBA/CaH mice, specified pathogen free, were obtained from the Animal Resources Centre (Perth, Australia), housed in a controlled environment (Microbiology Animal House, University of Western ECE INHIBITION IN VIRUS-INFECTED MOUSE LUNG Australia, Nedlands, Australia) and received food and water ad libitum. Mice were anaesthetized (60 mg.kg-1 pentobarbitone sodium, (i.p.)), and an osmotic minipump (Alzet 1007D; Alza Corporation, Palo Alto, CA, USA; 7 day duration) containing CGS 26303 or placebo (0.25 M NaHCO3) was implanted subcutaneously on the back immediately posterior to the scapulae. CGS 26303 was dissolved in 0.25 M NaHCO3 at 8.75 mg.mL-1 and was delivered at a rate of 0.5 mL.h-1 (~5 mg.kg-1.day-1). Respiratory tract virus stock and animal inoculation Influenza A/PR-8/34 virus was grown in the allantoic fluid of 10-day-old embryonated chicken eggs at 378C for 3 days as described previously . The allantoic fluid was harvested and contained 2.76106 mL-1 egg infectious doses (EID50) of virus as determined by the method of allantois-on-shell titration for infectivity . The virus stock was stored in 0.5-mL aliquots at -858C. Two days after implantation of osmotic minipumps, mice were anaesthetized with Penthrane (methoxyflurane, 1 mL added to a 500 mL-sealed container; Medical Developments, Melbourne, Australia) and groups of CGS 26303-treated animals and placebo-treated controls were intranasally inoculated with 15 mL of fluid containing 1,000 EID50 doses of influenza A virus. The remaining mice were sham-infected using a 15 mL solution of a 1:40 dilution of allantoic fluid from virus-free chicken eggs. On day six of the study (i.e. day four post-inoculation), mice were sacrificed by an overdose of pentobarbitone sodium (200 mg.kg-1, i.p; Rhone Merieux Australia Pty Ltd., Pinkemba, Australia). A 0.4-mL blood sample was taken to determine the levels of free CGS 26303 in the circulation. Plasma samples were centrifuged in Centrifree tubes (Amicon, Beverly, MA, USA) and the concentrations of CGS 26303 in the filtrates were measured using a neutral endopeptidase 24.11 inhibition assay. Lungs were blotted dry, weighed and prepared for determination of lung viral titres and extraction of ET. The trachea from each animal was carefully cleaned of adherent fat and connective tissue and prepared for autoradiography. Lung virus titres Lung tissues were homogenized in sterile saline with glass/glass tissue homogenizers, and the resulting suspension was clarified by centrifugation at 2,0006g for 5 min at 48C. Infectious virus was assayed by allantosis-onshell titration for infectivity as previously described . Briefly, 666 mm pieces of allantois-on-shell from 11day-old embryonated chicken eggs were incubated in sterile round bottom tubes containing 0.35 mL of Standard Medium (SM) and 25 mL aliquots of serial 10-fold dilutions of virus (10-2 to 10-6) in SM. Five replicates were used at each dilution. Tubes were sealed and placed in a working horizontal shaker in a 358C room for 48 h. The fluid from each tube was transferred to a haemagglutination tray and one drop of 10% washed goose red blood cells was added to each well. The trays were shaken and left to stand for 40 min. Positive haemagglutination indicated infection and the EID50 was calculated by the method of THOMSON . The composition of the SM (pH 7.0) was (in mM): NaCl 137, KCl 8, 93 CaCl2 7.2, MgCl2 0.52, glucose 1.7, acid-free gelatin 2.0 g.L-1, phenol red 2.5 mg.L-1, penicillin 5 UmL-1, streptomycin 5 mg.mL-1 and amphotericin B 12.5 ng.mL-1. Autoradiographic studies Tracheal tubes were submerged in Macrodex (6% dextran 70 in 5% glucose) and frozen by immersion in isopentane, quenched with liquid nitrogen. Serial transverse sections (10 mm) were cut at -208C and thaw-mounted onto gelatin/chromealum coated glass microscope slides. These sections were pre-incubated (2610 min) at 228C in a buffer (50 mM Tris-HCl, 100 mM NaCl, 0.25% bovine serum albumin, pH 7.4) containing the protease inhibitor phenylmethysulphonylfluoride (10 mM), and then in another buffer containing 0.2 nM 125I-ET-1 alone for 2.5 h (total binding) or in the presence of BQ-123 (selective ETA receptor ligand; 1 mM) or sarafotoxin S6c (selective ETB receptor ligand; 100 nM). Nonspecific binding was determined in the combined presence of BQ-123 (1 mM) and sarafotoxin S6c (100 nM). After 2.5 h, tissue sections were washed twice for 10 min in buffer, rinsed in distilled water and dried under a stream of cold dry air. Emulsion-coated cover slips (Kodak NTB-2; Eastman Kodak Company, Rochester, NY, USA) were attached to one end of the glass slides with cyanoacrylate adhesive and incubated for 3 days at 48C. The emulsion-coated coverslips were developed (Kodak Dektol, 1:1; Kodak Australasia Pty Ltd., Melbourne, Australia) for 3 min, rinsed for 15 s in dilute acetic acid (2%) containing hardener (Ilford Hypam; Ilford Imaging Australia, Melbourne, Australia) and fixer (Ilford Hypam, 1:4; Ilford Imaging Australia) for 2.75 min. Tissue sections were then stained for 30 s with Gill's double strength haematoxylin, dehydrated in ethanol, cleared in xylene and mounted (DePeX; BDH Laboratory Supplies, Kilsyth, Australia) for light microscopy. Autoradiographic grain densities were determined using a computer-assisted grain detection and counting system . A total of twelve slides were assessed (46total binding, 36BQ-123, 36sarafotoxin S6c and 26BQ-123 and sarafotoxin S6c) and each slide contained one tracheal ring from each of 32 mice studied (i.e. eight mice in each of the four groups). Four estimates of grain density were made per tracheal ring; three over the tracheal smooth muscle band and one over a nontissue area in the airway lumen. Thus, a total of 1,536 fields were analysed (12 slides632 sections64 fields). Autoradiographic grain densities are expressed as grains.1000 mm-2 and presented as the mean grain densitySEM. Extraction of endothelin from lung tissue Lungs were homogenized with glass/glass tissue homogenizers in a buffer containing 1M acetic acid and 1 mg.mL-1 pepstatin A at a ratio of 10 mL buffer.g wet weight-1 of tissue. Lung homogenates were then incubated in a boiling water bath for 10 min to inactivate proteolytic enzymes, cooled to 48C and centrifuged at 100,0006g for 20 min. The resulting supernatants were frozen and stored at -858C. ET was extracted from tissue supernatants as described previously . Briefly, C18 Sep-Pak cartridges (Waters, Milford, MA, USA) were pretreated with 5 mL 90% acetonitrile (ACN) in 1% trifluoroacetic acid (TFA) followed by 5 mL 25% ACN in 1% TFA. Samples (100 94 P.J. HENRY ET AL. Drugs Substances used included: 125I-ET-1, ET-1, BQ-123 (cyclo(D-Trp-D-Asp-L-Pro-D-Val-L-Leu)), sarafotoxin S6c (Auspep, Melbourne, Australia), CGS 26303 ((S)-2-biphenyl-4-yl-1- (1H-tetrazol-5-yl) ethylamino-methyl phosphoric acid) (Novartis Pharmaceuticals Corporation, Summit, NJ, USA), penicillin, streptomycin, amphotericin B, pepstatin A, phenylmethylsulphonylfluoride (Sigma Chemical Co., St Louis, MO, USA). Statistical analysis In the current study, four groups of mice were studied in a typical 262 factorial design. Thus, two-way analyses of variance (ANOVA) were used to determine virus- and CGS 26303-induced changes in ir-ET content and ETB receptor density. The Bonferroni correction was used for multiple comparisons. A p-value #0.05 was considered statistically significant. Grouped data are presented as meanSEM or mean (95% confidence interval (CI)). Results CGS 26303 plasma levels and infectious viral titres Following administration by osmotic minipump (5 mg.kg-1.day-1 for 6 days), the plasma levels of CGS 26303 in sham-inoculated mice (257 nM; 95% CI, 190± 357 nM) were not significantly different from levels measured in virus-inoculated mice (162 nM; 95% CI, 105±251 nM; NS). Thus, the plasma levels of CGS 26303 achieved by osmotic pump administration were not affected by coincident viral infection. Similarly, the levels of infectious virus in the lungs of placebo-treated mice (7.460.32 log EID50 per lung) were not significantly different from levels measured in CGS 26303-treated mice (6.930.19 log EID50 per lung; NS). Thus, presence of CGS 26303 had no significant effect on the levels of infectious influenza A virus present in the lungs 4 days post-inoculation. Immunoreactive endothelin content Ir-ET content in the lungs of virus-infected mice (202 39 pg.lung-1) was 210% higher than that measured in sham-infected mice (6618 pg.lung-1, p<0.05) (fig. 1). However, pretreatment with CGS 26303, significantly attenuated this virus-induced increase in the levels of ir-ET (p<0.05, 2-way ANOVA), such that ir-ET levels 300 * ir-ET pg lung-1 250 200 150 100 50 0 Virus Sham Treatment Fig. 1. ± Levels of immunoreactive-endothelin (ir-ET) in the lungs of influenza A virus-infected (Virus) and sham-infected mice (Sham) during coincident administration of CGS 26303 (h) or placebo (u). Data are presented as meanSEM (n=6±7). *: p<0.05. were only 75% higher in virus-infected mice (10914 pg.lung-1) than in sham-infected mice (6213 pg.lung-1) (fig. 1). Lung wet weights On average, lung wet weight was 455% greater in virus-infected mice (1605 mg) than in sham-infected mice (1103 mg; p<0.05) (fig. 2). However, in CGS 26303-treated mice, lung wet weight was increased by only 175% during viral infection (1356 mg in virusinfected lungs versus 1162 mg in sham-infected lungs; fig. 2) (p=0.001, compared with placebo treatment). EndothelinB receptor densities The levels of specific 125I-ET-1 binding on tracheal smooth muscle were similar in all four groups of mice (two-way ANOVA, table 1), although the relative proportions of ETA and ETB receptors differed between 0.20 Lung wet weight g mL of lung supernatant diluted with 100 mL 1% TFA and 200 mL 50% ACN) were then applied to pretreated C18 Sep-Pak cartridges. After sample application the cartridge was washed sequentially with 20 mL 25% ACN in 1% TFA, 10 mL H2O and 10 mL 50% methanol. Ir-ET was eluted with 16 mL 50% methanol in 4% acetic acid. Eluted samples were then frozen at -858C and dried under vacuum in a freeze drier unit (Model FD3; Dynavac, Melbourne, Australia). Dried samples were reconstituted in sample buffer supplied with the enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical Co., Ann Arbor, MI, USA) and the assay performed in accordance with the manufacturer's instructions. The antibodies used in this assay cross-react with ET-1, ET-2 and ET-3, but not with big-ET. * 0.15 0.10 0.05 0.00 Virus Sham Treatment Fig. 2. ± Lung wet weight of influenza A virus-infected (Virus) and sham-infected mice (Sham) during coincident administration of CGS 26303 (h) or placebo (u). Data are presented as meanSEM (n=8). *: p<0.05. ECE INHIBITION IN VIRUS-INFECTED MOUSE LUNG Table 1. ± Influence of CGS 26303 on virus-induced changes in the densities of endothelin (ET)A and ETB receptors on murine tracheal smooth muscle Treatment CGS 26303 No Yes No Yes ET receptor density Virus Total ETA ETB No No Yes Yes 95.87.6 91.17.9 99.78.7 10510 28.26.2 34.85.2 68.010 50.65.7 67.612 56.34.7 31.75.3 54.38.0 Data are shown as meanSEM (n=7±8). *: density of autoradiographic grains associated with specific 125I-ET-1 binding. groups (fig. 3, table 1). Tracheal smooth muscle from placebo-treated, sham-infected mice contained a greater proportion of ETB receptors (687% of specific ET receptors) than ETA receptors (327% of specific ET receptors) (fig. 3). However, as previously described [7, 8], tracheal smooth muscle from virus-infected mice contained significantly lower densities of ETB receptors (fig. 3). Administration of CGS 26303 had no significant effect on the density of ETB receptors in sham-infected mice (624% of specific ET receptors), but markedly attenuated the virus-induced reduction in ETB receptors. In mice that received CGS 26303, virus infection caused only a 176% reduction in ETB receptor density compared with the 518% reduction observed in placebotreated mice (p=0.01, fig. 3). Discussion Elevated levels of ir-ET within the airways and lung have been observed in many disorders of the lung, including respiratory tract viral infection . In the current study, virus-induced increases in ir-ET levels in the lung were markedly attenuated by CGS 26303, an ECE in- ETB receptor density % total 100 80 * 60 40 20 0 Virus Sham Treatment Fig. 3. ± Endothelin (ET)B receptor density as a percentage of total ET receptor density in tracheal smooth muscle from influenza A virusinfected (Virus) and sham-infected mice (Sham) during coincident administration of CGS 26303 (h) or placebo (u). ETB receptor densities were determined in quantitative autoradiographic studies using 125I-ET-1 and receptor-selective ligands. Data are presented as meanSEM (n=7±8). *: p=0.01. 95 hibitor that has recently been shown to inhibit ET-1 synthesis in cultured guinea-pig tracheal epithelial cells . The combination of these data indicate that virusassociated increases in ir-ET content were due, at least in part, to enhanced synthesis of ET by the airway epithelium, although recent studies indicate that tracheal smooth muscle cells can also express prepro-ET-1 and ECE-1 messenger ribonucleic acid (mRNA) . Although the underlying mechanism of virus-induced increases in ET content is unknown, an increased expression of ET1 mRNA is likely to be involved . Cytokines produced during respiratory tract viral infection , stimulated prepro-ET-1 mRNA expression and ET-1 release  as well as ECE-1 mRNA expression  in human cultured bronchial epithelial cells. Although virus-induced increases in ECE levels have yet to be demonstrated, examination of the structure of the promoters for the ECE-1a and ECE-1b gene suggest that it is the latter isoform that is most likely to be expressed in pathological states , such as respiratory tract viral infection. Elevated levels of ET-1 are associated with reduced ETB receptor densities in transplanted , congested  and virally-infected [4, 8] lungs. In the current study, CGS 26303 inhibited both the virus-induced increase in production of ir-ET and the reduction in ETB receptor density. One explanation for these findings is that viral infection increased the production of ET which then induced ETB receptor downregulation, a direct mechanism that is consistent with previously published studies showing that the ETB receptor is readily susceptible to desensitization in airway preparations [7, 9, 22]. Whether this CGS 26303-induced attenuation of virus-induced loss of ETB receptor density is translated into preserved ETB receptor-mediated contractile responses in tracheal smooth muscle must await additional studies. An alternate, or perhaps additional, mechanism that should be considered is that the increased production of ET during viral infection promoted the release of inflammatory cell cytokines and mediators, which in turn caused a reduction in ETB receptor density. Consistent with this latter indirect mechanism, ET-1 has demonstrable proinflammatory effects in the airways, including the influx of eosinophils in a murine model of allergic inflammation . In addition, various inflammatory cell cytokines, including interleukin (IL)-1 and tumour necrosis factor (TNF)-a, have been reported to modulate ET receptor levels [24, 25]. In this regard, it is interesting to note that CGS 26303 inhibited the virus-induced increase in lung wet weight, perhaps by blunting the pro-inflammatory effects of ET-1. However, the CGS 26303-induced reduction in virus-induced lung weight may be otherwise explained by considering haemodynamic influences, such as a reduction in ET-1-induced venoconstriction and the associated pulmonary congestion. The levels of infectious virus measured in the lungs of mice on day four post-inoculation were not significantly influenced by concomitant administration of CGS 26303 and, similarly, the plasma levels of CGS 26303 were not significantly affected by respiratory tract viral infection. Thus, it is unlikely that the observed changes in the levels of ir-ET and ETB receptor density in virus-infected mice treated with CGS 26303 (compared with placebo) were due to impaired growth of virus in the airways of CGS 26303-treated mice. Similarly, the differential effect of 96 P.J. HENRY ET AL. CGS 26303 on ir-ET and ETB receptor levels in virus- and sham-infected mice cannot be explained on the basis of differences in the plasma levels of CGS 26303. CGS 26303 is a potent inhibitor of neutral endopeptidase (NEP) 24.11 as well as ECE and thus due consideration must be given to the possibility that the effects observed in the current study may, at least in part, have resulted from NEP inhibition. NEP is present in the airway epithelium of several animal species, including humans [26, 27], and is thought to play a role in the catabolism of ET-1 [14, 28]. Thus, in the present study, CGS 26303induced inhibition of NEP may have had several effects. Firstly, NEP may well act as an "endothelinase" and thus inhibition of NEP by CGS 26303 might be expected to reduce the breakdown of ET-1 and thus increase its levels. However, compared to placebo-treated mice, the levels of ir-ET in CGS 26303-treated mice were either unchanged (in sham-infected mice) or significantly reduced (virusinfected mice), indicating that inhibition of the production of ET rather than prevention of its breakdown was the predominant functional effect of CGS 26303 in the current study. Secondly, inhibition of NEP by CGS 26303 may elevate the levels of other bioactive peptides, with unknown effects on the ET system. However, although NEP plays a role in the regulation of lung growth and maturation in foetal mice , the levels of NEP in the upper airways of adult mice are very low (G. Colasurdo, personal communication) in comparison to several other animal species. Indeed, although hitherto untested differences in the activity of NEP in mouse and rat trachea may explain, at least in part, the recently reported observation that parainfluenza-1 virus significantly attenuates ETB receptor density and function in mouse, but not in rat trachea . The higher activities of NEP in rat trachea may lead to greater catabolism of ET-1 and thereby protect the ETB receptor from downregulation. In summary, although further definitive studies are required, it appears that within the murine trachea the major influence of CGS 26303 is on ECE rather than NEP. The major finding of the current study was that CGS 26303 inhibited the virus-induced increase in immunoreactive endothelin content as well as the decrease in endothelinB receptor density. 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